Heat Shock Proteins (HSPs)
The hydrophobic regions of proteins are generally secluded, internal features in an aqueous environment. However, due to the high protein concentration within cells as well as fluctuations in charged molecules and physical parameters (“cellular stress”), the risk of exposure of hydrophobic regions is probably constant between synthesis, transport and function (Frydman J. 2001; Yam et al. 2005). Aside from compromised function, exposed hydrophobic regions cause protein aggregation, membrane damage and apoptosis.
Heat shock proteins (HSPs), also known as chaperones, bind to exposed hydrophobic regions to minimize such damage. HSPs are broadly conserved throughout the spectrum of life, frequently exhibiting 70% homology or greater in amino acid sequence between bacterial and human representatives (Bukau and Horwich 1998; Daugaard et al. 2007). In bacteria there is generally a single gene for HSP60, HSP70 and HSP90. Expression may be increased during conditions that unfold proteins such as fluctuations in temperature and ion concentration.
In higher eukaryotic organisms such as mammals, there are distinct isoforms of HSPs that have restricted subcellular location. For HSP70, there are isoforms for general activity in the cytoplasm (HSP70-8), endoplasmic reticulum (HSP70-5; grp78 or BiP) and mitochondrion (HSP70-9; mortalin). However, during potential cellular crisis periods such as anoxia, nutrient limitation, or ionic imbalance, the unfolding of proteins induces the expression of additional isoforms such as HSP70-1 (Scriven et al. 2007; Qian et al. 2006).
Broadly, HSP isoforms may be referred to as un-inducible (constitutive expression) or inducible (expression stimulated by cellular stress). The probability of HSP binding to appropriate polypeptide ligands inside the cell is influenced by ATP hydrolysis. The repeated loading of ATP, and release of ADP, cause allosteric changes in HSPs; these changes in conformation determine accessibility of the peptide binding domain to candidate “clients”. The binding and release of distorted proteins by HSPs may permit the clients to refold and continue function as cellular equilibrium is restored.
Some HSP family members (such as HSP90) also participate as facilitators, supporting the conformation of receptors and kinases for greater efficiency in their signaling activity or post-translational modification of other molecules (e.g. Bron et at. 2008). Alternatively, where protein distortion or fragmentation is beyond recovery, HSP family members participate in directing irreparably damaged proteins for further reduction into peptides (Young et al. 2004; Bukau et al. 2006). These peptides may ultimately end up on the surface of cells in association with molecules of the major histocompatibility complex (MHC) for inspection by cells of the immune system (Ishii et al. 1999; Binder et al. 2001).
However, even when released due to necrosis or other cellular trauma, HSPs retain their capability to stimulate immune responses to bound peptides and proteins even in the negligible presence of extracellular ATP/ADP (Henderson et al 2010a; Henderson et al 2010b; Suto and Srivastava 1995; Castellino et al 2000; Basu et at. 2001; Tobian et al 2004; Chen and Cao 2010)
HSP Directed Immunotherapy
The ability to harness the immunomodulatory capacity of HSPs is highly desirable (Binder 2008; Karapanagiotou et al 2009). Experimental evidence for artificial stimulation of the various immune responses is unequivocal, either when HSP complexes are enriched from diseased tissue or are prepared by genetic or chemical synthesis. However, the extrapolation of these observations to consistent clinically relevant results (i.e. reduced morbidity and recurrence free survival) remain elusive (Moroi et al. 2000; Vanaja et al. 2000).
The reason for the shortfall in expectations of HSP directed immunotherapy may include the possibility that contemporary protocols do not provide sufficient antigenic information to account for genetic variability within the population at risk (Davila et al. 2010; Jacobson 2004). Furthermore there will be different affinities of different proteins and peptides for different HSPs—only a small fraction of peptides may bind efficiently and these may out compete or exclude those necessary for a robust immune response (Flechtner et al. 2006).
To date, in vitro methods permit preparation of HSP70 (Bolhassani et al. 2008; Nishikawa et al., 2008) complexes with single peptides, chemically cross-linked or expressed in tandem with recombinant HSP70. Individually such preparations may not reflect natural conformations desirable for successful engagement of relevant receptors (Becker et al. 2002; Binder R J 2009). The recombinant approach may also be prohibitively expensive in the provision of comprehensive antigen coverage.
Stability and longevity may also be a limiting factor to clinical success of contemporary HSP immunotherapy methods; following vaccination, HSP-conjugates may not be sufficiently robust to establish contact with antigen presenting cells (APCs) due to susceptibility to serum peptidases (Micheilin et al. 2005).
Availability of vaccine material is an additional factor governing success of HSP immunotherapy: restricted sources of antigenic material may preclude a strong primary, sustained or anamnestic immune response. Inadequate amounts of starting material may be the most important limitation for HSP based immunotherapy. Restricted availability of HSP based immunotherapeutic material may be due to economically prohibitive number of required epitopes to provide individual or population wide coverage. Further, some important antigens may be difficult to produce in the laboratory. Such antigens include membrane proteins or those that require post translational translation (e.g. glycosylation: the addition of carbohydrate groups). Contemporary methodologies may also prone to reductive losses during the sequences of fractionation and preparation (e.g. during ADP chromatography). Where a patient's own tumor material is used as the source of HSP conjugate, obvious limitations exist predicated by the amount of starting source material. For example, a recent phase III trial incorporating HSP complexes enriched from individual patient tumors yielded largely unremarkable results. However, the study indicated that patients receiving repeated doses (predicated by larger tumors) had better parameters of immune response and median survival (Binder 2008). This important observation indicates that availability of material for sustained vaccination will be a factor in determining the success of HSP-based immunotherapy. This issue has been addressed by Katsanis, Graner and colleagues in which whole cancer cell lysates have been fractionated to produce chaperone rich lysates using free solution isoelectric focusing (FS-IEF) (Kislin et al. 2007; Bleifuss et al. 2008).
HSPs in Cancer Cells
HSPs are both cytoprotectants and powerful modulators of the immune system (Henderson et al. 2010c). However, as cytoprotectants, HSP expression is considerably over-extended in cancer cells where their function has been exploited to an extraordinary degree (Jäättelä 1995; Cappello et al 2003; Daugaard et al 2005; Rohde et al 2005; Sherman and Multhoff 2007). During oncogenesis, for example, overexpression of HSPs such as HSP90 provides structural support for constitutively active proteins that drive unregulated cell multiplication (Lewis et al. 2000; Broemer et al. 2004) Up-regulated HSPs may also promote survival and stall apoptosis within an otherwise prohibitively hostile environment characterized by anoxia and low nutrient availability (Powers et al 2009).
Over-expression of HSPs, as is typical of cancer, may also cause confusion and subversion of immune effectors directed against out of context expression of proteins permitting uninhibited cell division or survival in hostile environments (Chalmin et al. 2010; Su et al. 2010; Coelho et al. 2008). Due to the support and stabilisation necessary for continued function of membrane proteins, unlike normal cells, HSPs are found on the external surface of cancer cells (Graner et al. 2009; Cappello et al. 2008). (Horváth et al 2008). Consequently vesicular material released by cancer cells are also richly accessorised by HSPs (Broquet et al 2003; Lancaster and Febbraio 2005; Evdonin et al 2006; Mambula and Calderwood 2006).
Cumulatively, the above functions render cancer cells to become addicted to HSP over-expression: Without such increases, many cancer proteins would unravel and be directed toward degradation. Such losses would deny the cancer cell of important survival factors causing apoptosis and cell lysis. Indeed, many cancer therapies currently in development are depending upon the efficacy of HSP inhibitors (Banerji 2009; Powers et al. 2007; Powers et al. 2010; Davenport et al. 2010).
The use of cell derived vesicles (CDVs), such as exosomes for detecting biomarkers for diagnostic, therapy-related or prognostic methods to identify phenotypes is described in WO 2010/056337, the contents of which are specifically incorporated herein by reference.